Identification and Manipulation of Memory Engram Cells The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Liu, Xu, Steve Ramirez, Roger L. Redondo, and Susumu Tonegawa. “Identification and Manipulation of Memory Engram Cells.” Cold Spring Harbor Symposia on Quantitative Biology 79 (2014): 59–65. As Published http://dx.doi.org/10.1101/sqb.2014.79.024901 Publisher Cold Spring Harbor Laboratory Press Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/106800 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ Identification and Manipulation of Memory Engram Cells Xu Liu1,2,3, Steve Ramirez1, Roger L Redondo1,2, Susumu Tonegawa1,2 1RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. 2Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. 3Current address: Department of Neurobiology, Northwestern University, Evanston, IL 60208, U.S.A. Running Head: Memory Engram Cells Corresponding author: Prof. Susumu Tonegawa 43 Vassar St., 46-5285, Cambridge, MA 02139, USA Phone: (617) 253-6459 Fax: (617) 258-6893 Email: [email protected] Abstract How memories are formed and stored in the brain remains a fascinating question in neuroscience. Here we discuss the memory engram theory, our recent attempt to identify and manipulate memory engram cells in the brain with optogenetics, and how these methods are used to address questions such as how false memory is formed, and how the valence of a memory can be changed in the brain. 2 How and where memory is stored in the brain network is one of the fundamental questions in brain and cognitive sciences. At the onset of the 20th century, a German biologist Richard Semon proposed the engram theory of memory (Semon 1923), but the theory was nearly completely ignored by his contemporary and subsequent brain researchers, until Daniel Schactor, James Eich, and Endel Tulving revived the theory in the late 1970s (Schacter et al. 1978). Semon’s memory engram theory was built on two fundamental postulates termed the “Law of Engraphy” and the “Law of Ecphory” for memory storage and memory retrieval, respectively. The law of Engraphy posits: “All simultaneous excitations (derived from experience)…with in our organisms form a connected simultaneous complex of excitations which, as such, acts engraphically, that is to say leaves behind it a connected, and to that extent, unified engram-complex” (Semon 1923). The Law of Ecphory on the other hand posits: “The partial return of an energetic situation which has fixed itself engraphically acts in an ecphoric sense upon a simultaneous engram-complex” (Semon 1923). Semon’s conceptualizations of the memory process were novel at his time, and were remarkably predictive of the contemporary concepts of memory storage and retrieval. For instance, Semon’s memory retrieval process contained the concept of “pattern completion,” which was advanced years later (Marr 1970; Nakazawa et al. 2003; Leutgeb et al. 2004). However, Semon did not elaborate the biological basis of the “simultaneous excitations” nor “a connected, unified engram-complex.” This is not surprising considering that his theory was put forward nearly a century before the development of molecular, cellular, and genetic biology, and sophisticated imaging and electrophysiological technologies for the analysis of the nervous system. Incorporating the current knowledge about neurons, synaptic connections, and neuronal circuits, Semon’s Engram Theory of Memory can be rephrased as follows: When a subject undergoes or 3 encounters and episode, a set of selected stimuli from the experience or episode activate populations of neurons to induce enduring physical and/or chemical changes (engrams) in them and their connections, each contributing to the storage of memory. Subsequently, when a part of the original stimuli returns, these cells (engram cells) are reactivated to evoke the recall of the specific memory. A half-century after Semon’s book was published, Karl Lashley pioneered a systemic hunt for engram cells in the rodent brain by introducing lesions of varying sizes into different areas of the cerebral cortex, attempting to find an engram for a maze task. However, Lashley found that memory was impaired in many of these lesioned animals, and the severity of the impairments was proportional to the sizes of the lesions. On the basis of these findings, Lashley concluded that the engrams for maze-resolving memory are spread throughout the cerebral cortex with no obvious localization (Mass Action Principle) (Lashley 1950). However, soon after Lashley’s study, Wilder Penfield and Theodor Rasmussen obtained the first evidence suggesting that the engrams of episodic memories are stored in the medial temporal lobes (MTLs) (Penfield and Rasmussen 1950). This chance finding was supported several years later by William Scoville and Brenda Milner, who discovered that a patient H.M., who lost a large portion of his MTLs due to surgery, had severe anterograde amnesia for episodes as well as a graded retrograde amnesia. These studies were consistent with the notion that episodic memories are stored in the MTLs. As to the nature of memory engrams—enduring physical and chemical changes induced by learning—the guiding hypothesis has been Donald Hebb’s theory, which posits that neurons encoding memory stimuli undergo enduring strengthening of some of their synapses through their co-activation with presynaptic cells: neurons that “fire together wire together” (Hebb 1949). Starting with Tim Bliss and Terje Lomo’s discovery of long-term potentiation (Bliss and Lomo 4 1973), which supports Hebb’s hypothesis, a large amount of studies have been directed to the characterization of LTP and other facets of synaptic plasticity, and their potential role in learning and memory. However, none of these studies could link these activity-dependent alterations of synapses and neurons directly to engram cells which are activated by specific learning and whose reactivation by the specific recall cues elicited behavioral responses. In this review, we shall outline our recent attempt to identify memory engram cells, and to manipulate them by optogenetics in order to investigate several thus far unresolved issues associated with episodic memory. Optogenetic Activation of Memory Engram Cells Previous studies have linked selected neuronal populations with particular memory events by correlational evidence (Reijmers et al. 2007) and loss-of-function evidence (Zhou et al. 2009; Han et al. 2009), but a critical piece of evidence was largely missing. The most direct evidence of engram cells should come from gain-of-function experiments, where a population of neurons that are considered responsible for a particular memory are selectively labeled and activated artificially to mimic their natural activity. If such manipulation causes the recall of that memory, then this provides evidence that the selected population of neurons is sufficient for the memory, thus argues the selected neuronal population is the neuronal basis for the engram of this particular memory (Martin and Morris 2002). However, this type of gain-of-function experiments are technically challenging, as one has to be able to correctly isolate the neurons involved in one particular memory from their seemingly indistinguishable neighbors and activate 5 them with proper spatial and temporal precision. Recent advances in technology such as optogenetics (Goshen 2014; Fenno et al. 2011) enabled such experiments. To achieve this goal, we combined activity-dependent, drug-regulatable expression system with optogenetics (Liu et al. 2012). We used a transgenic mouse model where the artificial tetracycline transactivator (tTA), which can be blocked by doxycycline (Dox), is driven by the promoter of immediate early gene (IEG) c-fos (Reijmers et al. 2007). The activity-dependency of c-fos promoter poses a natural spatial constrain on the identities of the neurons that can be labeled, reflecting the normal biological selection process of the brain during memory formation, while the Dox-dependency of the system poses an artificial temporal constrain as to when these neurons can be labeled, which can be controlled by the experimenters. With these two constrains, the down stream effector of tTA can express selectively in neurons that are active during a particular behavior episode, only if the animals are off Dox diet. Using this system, we expressed channelrhodopsin-2 (ChR2) delivered by a viral vector AAV-TRE-ChR2-EYFP targeting the dentate gyrus (DG) of the hippocampus and implanted optical fibers right above the infected areas (Fig. 1A). These animals were habituated in one context A with light stimulation while on Dox, then taken off Dox and fear conditioned in context B, where DG neurons active during the formation of this context-fear association memory were labeled by ChR2 (Fig. 1B), after which they were put back on Dox diet to stop further labeling, and tested again in context A by light stimulation of the labeled neurons (Fig. 1C). Although light had no effect on the test subjects in context A before training, these animals showed reversible, light-dependent freezing in context A after training (Fig. 1D),